Inhibition Of Protein Kinase D By The Small Molecule CID755673 Promotes Mouse And Human Embryonic Stem Cell Self-renewal

Mouse embryonic stem cells(m ESCs)derived from the inner cell mass of blastocyst have two characteristics in vitro,self-renewal and pluripotency.The self-renewal refers to the ability of m ESCs to continuously proliferate under specific conditions in vitro without differentiation and pluripotency,meaning that m ESCs have ability to differentiate into ectoderm,mesoderm,and endoderm.Researchers isolated ESCs from mice in 1981.This is also the first ESC line successfully established in vitro,which extends people's understanding of m ESCs.After 7 years,the human embryonic stem cell(h ESCs)line was successfully established,prompting the basic research of h ESCs.The application of h ESCs in the field of life medicine has become a hot topic of concern to the majority of researchers.m ESCs and h ESCs have similar characteristics while also harbor many differences,such as the culture conditions and cell morphology.Notably,only mouse and rat ESCs have ability to generate germline chimera animals in vivo.Researchers have been working hard to develop suitable media to maintain the growth of ESCs for different animals in vitro.Based on this problem,we will try to find new mechanisms for maintain the self-renewal of ESCs,and then want to optimize the current culture conditions to maintain ESCs of different animals.First,we used a small molecule library to do screening and found that 46 C m ESCs treated with CID755673(CID)exhibited an undifferentiated phenotype.At the same time,alkaline phosphatase staining(AP),immunofluorescence and quantitative real-time(q RT-PCR)were performed and showed that CID-treated m ESCs had strong AP activity and high expression of pluripotency genes,Oct4 and Nanog,suggesting that CID can promote the self-renewal of m ESCs.Next,by adding different concentrations of CID to m ESC culture basal medium without LIF,a self-renewal-promoting cytokine,we found tha t m ESCs have the best phenotype and AP activity under 10 ?M CID.However,these m ESCs gradually differentiated after third passages,suggesting that CID can only maintain the short-term self-renewal of m ESCs in in vitro.In order to investigate whether CID can maintain the stemness of m ESCs for a long time with other small molecules,we set up two experimental groups including CID plus PD0325901(PD)and CID plus CHIR99021(CHIR).The results showed that 46 C m ESCs have strong AP activity,high expression of Oct4,Sox2 and Nanog and can be passaged multiple times in the presence of CID and PD.We therefore named this condition as PC.PC-maintained m ESCs can form embryoid bodies and then can differentiate into cells of the endoderm,mesoderm and ectoderm.In addition,after being injected into the blastocyst,PC-treated m ESCs have ability to generate a germ-line competent chimeric mouse.Overall,these data indicate that PC can maintain the pluripotency of m ESCs in vitro.CID is a specific inhibitor of the protein kinase D(PKD)family,which contains three members PKD1,PKD2 and PKD3.PKD genes are involved in regulating many cell biological functions,such as the integrity of the Golgi complex and protein secretion,cell apoptosis and proliferation,oxidative stress and inflammation and the development of heart disease and malignant tumors.In order to determine which PKD is downstream of CID to promote 46 C m ESC self-renewal,we knocked down PKD1,PKD2 and PK D3 in 46 C m ESCs,respectively.Down-regulation of PKD alone is not enough to maintain the identity of m ESCs,indicating that the function of the PKD family genes may be complementary.Therefore,CRISPR/CAS9-mediated DNA double-strand mutation was used to knock out PKD gene expression in 46 C m ESCs together.Compared with wild-type cells,the rate of differentiation of ESCs with three PKD genes knocked out delayed,indicating that CID promotes the self-renewal of m ESCs by simultaneously inhibiting the activity of three PKD members.Next,in order to explore the molecular mechanism by which CID promotes the self-renewal of m ESCs,we used high-throughput sequencing technology to analyze the changes of gene expression in m ESCs treated with PD or PC.Compared with the PD group,the expression of 613 genes was up-regulated and the expression of 980 genes was down-regulated in PC-treated cells.Combined with the analysis of functional enrichment of bioinformatics,genes associated with the regulation of stem cell pluripotency were identified.The q RT-PCR method was further performed to verify their expression and two gene,Pik3r3 and Id2,were identified.However,in the presence of PC,cells in which Id2 was suppressed still had typical ESCs morphology,indicating that Id2 is not a key target gene of CID.Therefore,we chose another candidate gene,Pik3r3.Previous studies have shown that overexpression of Pik3r3 can increase the phosphorylation of AK T,while knockdown of Pik3r3 decreases the phosphorylation of AKT.we also verified these results.In addition,western blot detection showed that CID increased the phosphorylation level of AK T.We then added a specific small molecule inhibitor of PI3K/AKT signaling pathway,LY294002(LY294),to the CID-containing medium.Addition of LY294 did not weaken the ability of PC to maintain the self-renewal of m ESCs,while differentiation appeared on the edge of some cell clones.These findings collectively indicate that the PI3K/AKT signaling pathway can partially mediate the ability of CID to promote the self-renewal of m ESCs.Finally,in order to determine whether CID can promote the self-renewal of h ESCs,CID was added to h ESC basal medium without addition of self-renewal cytokines,we found that compared with the untreated control group,CID-treated cells had stronger AP activity and higher expression of self-renewal marker genes OCT4 and NANOG.At the same time,CID also works by inhibiting three PKD genes together,because knockdown of PKD1,PKD2 or PKD3 alone could not maintain the undifferentiated state of the h ESCs,while downregulation of three PKD genes can slow down the h ESC differentiation.These cells have a strong AP activity,while addition of LY294 greatly reduced the self-renewal ability of CID in h ESCs.These data together shows that the self-renewal-promoting effect of CID is conversed in h ESCs.In summary,we reported a new condition for maintaining the self-renewal and pluripotency of ESCs in vitro through addition of small molecule CID,and further explored the downstream molecular mechanism.Meanwhile,we demonstrated that the role of the CID in mouse and human ESCs is conserved.These findings not only extend the understanding of the regulatory network of pluripotency in ESCs,but also provide clues for improving the culture conditions of pluripotent stem cells in vitro,which will promote stem cell basic and applied research in future.